International Journal of Pharmacy

Ghousia, et al. Int J Pharm 2013; 3(4): 655-662

ISSN 2249-1848

International Journal of Pharmacy Journal Homepage: http://www.pharmascholars.com

Research Article

CODEN: IJPNL6

HAEMATOLOGICAL EFFECTS AND SAFETY BEHAVIOUR OF 20 kDa PEGINTERFERON ALPHA-2A (UNIPEG®) IN HEALTHY HUMAN SUBJECTS *Ghousia. Saba1, Javeid. Iqbal1, Tasneem. Ahmad2, Ayaz Ali Khan1 1

Faculty of Pharmacy, Hamdard University, Karachi, Pakistan; 2Pharma Professional Services, A-93, Ettawah Society, Scheme 33, Karachi 7540, Pakistan *Corresponding author e-mail: [email protected] ABSTRACT 20 kDa pegylated interferon alpha-2a (UNIPEG®) manufactured through the pegylation of E.coli-derived interferon alpha-2a. The hematological effects of Unipeg® were studied after a single subcutaneous dose of 180 µg (protein content) in ten healthy human subjects. Vital signs, adverse events and lab tests (hematological and biochemical) were monitored for safety analysis. Clinical laboratory tests results before and after drug administration were processed for any statistically significant change by computing the ANOVA (F-value) and performing t-tests. Statistically significant decrease in neutrophils, absolute neutrophil count, total white blood cells count and platelets count were observed but most of these were not clinically significant. Transient leucopenia and leucopenia was reported for 10% and 20% subjects, respectively, transient thrombocytopenia in 10% subjects and an increase in ALT levels above normal ranges in 30% of subjects were observed while no serious adverse effect was reported. The hematological effects were found similar to those of reported in literature for unmodified IFN-alpha-2a and other pegylated IFN-alpha-2a products generally used in therapy. Key words: PEG-IFN- alpha

2a, 20-kDa, Unipeg, Pegylated interferon alfa-2a, safety.

INTRODUCTION The 20 kDa pegylated interferon alpha-2a (PEG(20kDa)-IFN-alpha-2a) is a covalent conjugate of the protein interferon alpha-2a (IFN-alpha-2a); produced by recombinant DNA technology in Escherichia coli, with a single stranded polyethylene glycol (PEG) molecule (20 kDa). [1] Interferon alpha2a is a naturally occurring glycoprotein which belongs to the class of cytokines and mainly produced in the body by leukocytes. It plays an important role as immune stimulant or in the up gradation of immune system against viral infections and tumor cells.[2] It has antiviral, anti-proliferative and immunoregulatory properties. [3] This IFN-alpha2a is susceptible for enzyme degradation and rapid clearance. Due to this property, IFN needs to be administered frequently; this leads to wide fluctuations in blood levels and interference in sustained therapeutic response. In addition, the varying IFN peak levels elicit more adverse effects.

[4]

These disadvantages limit its clinical usage. But, with the advent of pegylation in 1970, it was found that chemical modification of proteins can improve their therapeutic use; with added convenience of once-weekly dosing with no novel toxicities.[4] PEG optimizes the action of ‎the IFNs by decreasing clearance rates, thereby allowing serum concentrations to remain constant over ‎the dosing period and hence enhance efficacy. [5][6][7] ‎ Polyethylene glycol (PEG) is a polymer consists of repeating ethylene oxide units and two terminal hydroxyl groups. Due to its production process PEG is available as a mixture of molecules with a variation of chain length, this phenomenon is termed polydispersity. These mixtures are usually classified by their average molecular weight, a value that may vary between several hundred to several thousand Daltons Both linear and branched PEG molecules are available.[8] The PEG used in study drug (UNIPEG; manufactured by Getz Pharma (Pvt) Ltd, Karachi,

655

Ghousia, et al. Int J Pharm 2013; 3(4): 655-662 Pakistan) has a limited polydispersity of 20,000±2,000. Pegylation of IFN-alpha-2a optimizes its pharmacological activity; such that the efficacy is enhanced, while adverse effects, antigenicity and immunogenicity are minimized and less frequent dosing is required; which enhanced the patients’ compliance. [4][9] Change in PEG chains (either linear or branched) attached to the protein molecule; results in pegylated interferons (IFN-alpha-2a) with different chemical properties, molecular weight, pharmacokinetic and pharmacodynamic properties.[4][8] The PEG-IFNalpha-2a containing PEG chains of various chemical structures, size and molecular weight are commercially available in different countries of the world; e.g. 40kDa “Pegasys” (by Hoffmann-La Roch), 12kDa “PegIntron” (by Schering-Plough), 20 kDa, “Reiferon Retard®” (by Rhein Minapharma).[10][11][12] But, all these pegylated interferon therapies result in hematological cell suppression and causes cytopenia (thrombocytopenia, leucopenia, anemia, neutropenia). The aim of this study is to evaluate the hematological effects of locally manufactured PEG(20kDa)-IFN-alpha-2a in healthy human subjects. MATERIAL AND METHOD Investigational drug product: Each one ml ampoule of the Investigational drug UNIPEG® manufactured by Getz Pharma Pvt Ltd. Karachi, Pakistan contained PEG(20kDa)-IFN-alpha-2a 180μg protein. Besides the active ingredient the product also contained sodium chloride, acetic acid, sodium acetate, benzyl alcohol, tween 80, and water for injection as excipients. Subjects: The study was conducted on ten healthy human volunteers with due approval of Independent Ethics Committee in compliance to the Declaration of Helsinkiand ICH-GCP guidelines. All volunteers gave their free written informed consent prior to participation. For participation in the study only those individuals were considered who had a BMI of 18-26 Kg/m2, age: 18-45 years, healthy with no history of chronic diseases, did not suffer in any acute illness in the previous 30 days, had no untoward symptoms or signs revealed through physical examination and laboratory tests, and were negative to HIV and hepatitis B and C virus. Subjects were not included if they had received the treatment with interferon (IFN) with in last one year or had donated blood in the previous 2 months. Individuals with low blood counts and hematology results outside the normal range were not included. Absolute Neutrophil Count (ANC)

ISSN 2249-1848 less than 1500/mm3 and platelet count less than 50,000/ mm3 were exclusion criteria. Study Design: An open label, single dose study was conducted on ten healthy volunteers. 180mcg/ml of Unipeg® was injected in abdominal skin of the human volunteers. During the study, participants remained in the clinical facility for first 24 hours after the injection under strict medical supervision, then subjects were discharged and they visited the clinical facility at 36 hours then every 24 hours for physical check-up, vital signs and adverse event monitoring until 156 hours. Safety was monitored by complete physical and medical examination with clinical laboratory testing and scheduled vital signs surveillance. Clinical laboratory testing included hematological (hemoglobin, hematocrit, platelet, and total and differential leukocyte counts) and biochemical (transaminases) determinations as safety variables. Hemoglobin, ALT, Total WBC, Neutrophile % and absolute neutrophil count were conducted at screening and the values were taken as base line. On the fifth and 16th day after drug administration the tests were repeated and the results were compared with the base line values. Paracetamol was given orally after observing the raise in body temperature after dosing and every 6 hours thereafter up to 24 hours or more if needed. The Clinical Investigator closely monitored the subjects for adverse events (AE) and took all necessary actions in the best interest of subjects. Data Analysis: Vital signs and Clinical lab tests results before drug administration and afterwards were processed for evaluation of any statistically significant change by computing the ANOVA (Fvalue) and performing t-tests. Simple statistics were employed in the computation, in summary the statistical formulas employed are: Mean

SEM

Variance ratio

In ANOVA F is determined by

Percentage of volunteers suffered in different adverse events was also calculated.

656

Ghousia, et al. Int J Pharm 2013; 3(4): 655-662 RESULTS AND DISCUSSION Laboratories Abnormalities or Hematological Effects: Interferon alpha 2a (IFN-alpha-2a) exerts anti-proliferative effect on many cell types. These properties are used for treatment of chronic myeloporliferative and lympho-porliferative disease. But, this also results in hematological cell suppression or undesirable effects. IFN-alpha-2a therapy causes cytopenia (thrombocytopenia, leucopenia, anemia, neutropenia) and its main mechanism is seems to be bone marrow suppression especially for pleuripotent progenitor cell of all lineage which cannot be counteracted by the production of endogenous hematopoietic growth factor. IFN-alpha-2a therapy may also results in immune mediated hematological toxicity and capillary sequestration of platelets and WBC and seems to be the additional causes for thrombocytopenia and leucopenia. [13][14] Usually a normal complete blood count is recovered on discontinuation or dose modification of IFN-alpha-2a therapy. In this study the level of significance was determined for laboratory tests performed at screening, on day five and sixteen after single dosing; given in table 2. Neutropenia: In the present study after single dose administration of Unipeg® (PEG (20kDa)-IFNalpha-2a) in healthy volunteers, ANC remained above 1500/mm3 and no neutropenia was identified (shown in figure 1). While, with PEG(40kDa)-IFNalpha-2a in healthy volunteers; neutropenia was reported in 5% volunteers in a single study submitted in BLA 103964 by Hoffmann-La Roche. In this study, 180 mcg dose of PEG(40kDa)-IFN-alpha-2a was administered either subcutaneously or intravenously.[15] Marcellin et al. reported that neutropenia was the most common laboratory abnormality leading to dose modification during treatment with PEG(40kDa)-IFN-alpha-2a in patients. [16] Neutropenia after PEG(40kDa)-IFNalpha-2a administration in patients has been reported from 17 to 47.5%. [17][18][19] Decrease in Neutrophil Count: In present work it was observed that although single dose of PEG (20kDa)-IFN-alpha-2a did not cause neutropenia in any patient however, a decrease in neutrophil count from baseline values were identified in 100 % (10) volunteers but all these were within normal limits (i.e. 40-78%) with recovering trend towards their baseline values (shown in figure 2). In a single dose study of PEG(40kDa)-IFN-alpha-2a a decrease in neutrophil counts has also been reported in 50% of volunteers. In this study it was observed that thirty subjects had a total of 109 abnormalities, the majority

ISSN 2249-1848 which were decrease neutrophil counts (50%) and white blood cell count (30%). [20] Decrease in neutrophil count have also been reported in 17% volunteers for a PEG(40kDa)-IFN-alpha-2a study in healthy volunteers while evaluating its safety at 90, 180, and 270 µg subcutaneous dose.[15] Thrombocytopenia: Thrombocytopenia mainly causes the dose modification of interferon alpha 2a therapy in patients. García-García et al identified thrombocytopenia as most frequent event in healthy subjects (i.e. in 56.3% volunteers; platelets counts less then 150X109 cells/L) after PEG(40kDa)-IFNalpha-2a single dose injection. [21] In current study one case of transient thrombocytopenia and one case of very mild thrombocytopenis was observed. Taha et al studied the Reiferon Retard® in chronic hepatitis C (CHC) patient and reported that 12.15% patients suffered with thrombocytopenia during 24 to 48 weeks treatment period.[17] Thrombocytopenia has been observed in studies of PEG(40kDa)-IFN-alpha2a in different conditions e.g. renal cell carcinomas, chronic hepatitis C, HIV and hepatitis B in different percentages ranging from 6% to 17.2%. [16][[17][18][19][23][24][25][26] Marcellin et al. also reported thrombocytopenia and identified it as the major cause of dose modification for PEG(40kDa)-IFN-alpha2a.[16] However, in this present study after single dose of PEG(20kDa)-IFN-alpha-2a in healthy patients only one (10%) case of transient thrombocytopenia was observed with recovering trend and two cases (20%) of very mild thrombocytopenia were reported. Transient decrease in platelets count have also been reported in 33% (4) volunteers at a dose of 180 µg of PEG(40kDa)-IFN-alpha-2a in healthy volunteers while evaluating its safety at 90, 180, and 270 µg subcutaneous dose.[15] Serum glutamic-pyruvic transaminase /Alanine transaminase (SGPT/ALT): In our current study on day five, an increase from 5.5% to 108.69% in SGPT from baseline values was found in seven volunteers. But all these were within normal limits except for three volunteers (30%) in which SGPT raised above the normal value (above 41U/L) that is 48, 60 and 61 U/L. Out of these three; two subjects recovered on day sixteen of drug administration while one still had raised SGPT (64U/L). this indicates a transient elevation of ALT which returned to normal. GarcíaGarcía et al has also reported the increased transaminases (>100 UI) in 62.5% (n=10 out of 16) volunteers after single subcutaneous dose of PEG (40kDa)-IFN-alpha-2a. [25] Zeuzem et al. studied the PEG (40kDa)-IFN-alpha-2a plus ribavirin treatment in chronic hepatitis C patients with normal ALT levels compared to the patients with no treatment. He

657

Ghousia, et al. Int J Pharm 2013; 3(4): 655-662 concluded that transient elevations in ALT activity in treated and control patients during the study. No severe flares of ALT activity were associated with treatment, and the benefit-risk ratio was positive. [26] Absolute neutrophil count (ANC): In the present study a decrease in absolute neutrophil count (ANC) was observed in 100% or all volunteers. It (ANC) dropped in a range of 24.42% to 75.98% on day five. But, ANC remained above 1500/mm3. On day sixteen, recovering trend towards baseline value was observed for ANC in all volunteers. In seven volunteers, at follow-up; drop in ANC was 2.04% to 24.97% while it was 42.58%, 52.65% and 68.98% in three volunteers respectively. Although, the drop was marked but, no clinically significant neutropenia was reported. Varunok et al evaluated pharmacokinetics, user handling, and tolerability of PEG (40kDa)-IFNalpha-2a delivered via a disposable auto injector device in 50 healthy volunteers; all subjects received one 180 μg dose. He found a decrease in neutrophil counts in 50% patient that definitely have been resulted in decrease of ANC. [20] Taha et al also observed neutropenia in 17.76% patients during treatment with Reiferon Retard® (PEG (20kDa)-IFNalpha-2a) which indicates a decrease in absolute neutrophil count also. [17] Leucopenia or decrease in total leucocytes count: In the current study with PEG (20kDa)-IFN-alpha-2a a decrease in leucocytes count was recorded for all volunteers (100%). On day five after drug administration WBC count drop from 27.4% to ‎60.97%. But, all these were within normal limits of WBC count (i.e. 4.0-‎‎11.0X109/L); except for three volunteers who had leucopenia with a drop ‎of 33.33%, 47.69% and 51.61% respectively. The recovering trend towards ‎baseline value of TLC was

ISSN 2249-1848 observed on day sixteen of drug ‎administration; except for two volunteers (20%) who still had leucopenia (shown in figure 3).‎ García-García et al also reported leucopenia 87.5% volunteers after a single dose of 180 mcg.[25] Decrease in WBC count have also been reported in 67% (n=8 out of 12) volunteers at a dose of 180 µg of PEG (40kDa)-IFNalpha-2a in healthy volunteers while evaluating its safety at 90, 180, and 270 µg subcutaneous dose. [15] Serious adverse events and death: In current study no serious adverse event or death has occurred. Similarly, no death and serious adverse event have been reported in any other single dose study of PEG (40kDa)-IFN-alpha-2a.[15][17][25] CONCLUSION PEG (20kDa)-IFN-alpha-2a (Unipeg®) ‎administered subcutaneously as a single dose, shows hematological effects similar to other IFN-alpha-2a products. Although a decrease in neutrophil count, WBC, Absolute neutrophil count and platelets count was observed, none indicated neutropenia or clinically significant abnormality. ACKNOWLEDGEMENT I pay thanks to all those who helped and supported me in this work and specially to Dr. Ben Rademaker & Mr. Jaffer Bin Baqar for providing manuscript review. FUNDING Research work funding was provided by an unrestricted academic grant by Getz Pharma (Pvt) Ltd, Karachi, Pakistan.

Table 1: Demographic and baseline characteristics of subjects Gender Age (years) Height (Cm) Weight (kg) BMI (Kg/m2) Race Mean Hb(gm) Mean WBC Mean Absolute Neutrophil Count Mean Platelets Count

All Male 25.2 ± 5.33 (20-32) 166(160-170) 59.60 ± 7.71 (60-74) 21.65 ± 2.71 (18.0-25.5) South Asians (Pakistani) 14.45 8X109/L 4500/mm3 250X109/L

658


ISSN 2249-1848

Table 2: Analysis of variance and p-value for clinical lab results Parameter

During the study (on fifth day of drug administration) F-value p value Sig at 1% Sig at 5% level of level of significance significa nce 0.6164 0.4426 Ns Ns

Hemoglobin (14-18g/dl) Total WBC 21.718 10^9 /l Neutrophils% 5.4706 Absolute 15.7624 neutrophils count Platelates 10^9 9.477 /l ALT 2.8238 RBC 0.0592 PCV% 0.7848 MCV 0.2148 Lymphocytes 9.4967 (20-45%) Monocytes (15.1984 10 %) Eosinophil (16.21547 5%) Ns: Not significant

After the study on follow up (on day 16 of drug administration) F-value p- value Sig at 1% Sig at 5% level of level of significanc significa e nce 1.5480 0.229 ns ns

0.00019

√

√

7.605222

0.0129

√

√

0.0311 0.00089

Ns √

√ √

0.411673 5.12642

0.529 0.036

Ns Ns

Ns √

0.0064

√

√

0.3008

0.590

Ns

Ns

0.110 0.8105 0.387359 0.64858 0.00643

Ns Ns Ns Ns √

Ns Ns Ns Ns √

-0.2733 1.0463 0.1117 1.013

-0.607 0.319 0.742 0.327524

Ns Ns Ns ns ns

Ns Ns Ns Ns Ns

0.03501

Ns

√

3.5095

0.077349

ns

Ns

0.02263

ns

√

1.9625

0.178251

ns

Ns

Figure 11: Mean neutrophils percentage observed in the study

659


ISSN 2249-1848

Figure 2: Change in absolute neutrophil count in all volunteers

Figure 3: Mean WBC count observed in the study

660


ISSN 2249-1848

REFERENCES 1. 2. 3. 4. 5. 6. 7.

8. 9.

10. 11.

12. 13.

14.

15.

16.

17.

18.

19.

20.

21.

Unipeg [Package Insert]. Karachi, Pakistan : Getz Pharma (Pvt) Ltd, 2010. Gilman Goodman, Hardman Joel G and Limbird Lee E, editiors. Goodman and Gilman's The pharmacological basis of therapeutics. 11th ed. New York: McGraw-Hill; 2006. pp. 1332-1334. Sweetman Sean C, editor. Martindale the complete drug reference. 36th ed. Chicago: Pharmaceutical Press; 2009. pp. 885-891. Rajender K Reddy, Marlene W. Modi, Simon Pedder. Use of peginterferon alfa-2a (40 kDa) (pegasys) for the treatment of hepatitis C. Advanced Drug Delivery Reviews. 2002:5:571-86. P, Caliceti. Pharmacokinetics of pegylated interferon: what is misleading? Digestive Liver Disease. November 2004:36:S334-9. Stefan Zeuzem, Christoph Welsch and Eva Herrmann. Pharmacokinetics of peginterferons. Seminars in Liver Disease. 2003;23:23-28. Paul J. Pockros, Robert Carithers, Paul Desmond, Daniel Dhumeaux, Michael W. Fried, Patrick Marcellin, et al.Efficacy and safety of two-dose regimens of peginterferon alpha-2a compared with interferon alpha-2a in chronic hepatitis C: A multicenter, randomized controlled trial. American Journal of Gastroenterology. 2004;99(7):1293-1305. Michael J Grace and David Cutler. Pegylating IFNs at His-34 improves the in vitro antiviral activity through the JAK/STAT pathway. Antiviral Chemistry & Chemotherapy.2004;15(6):287–97. Sebastian, Bailon Pascal and Vallejo, Palleroni Alicia.European Patent Specification for interferon Conjugates. European patent EP 0809996 B1 European, 2003 April, 2. http://www.patentlens.net/patentlens/patents.html?patnums=EP_0809996_B1&language=&. GR, Foster. Pegylated interferon with ribavirin therapy for chronic infection with the hepatitis C virus. Expert opinion Pharmacotherapy. 2003 May;4 (5):685-91. G Esmata and S. Abdel Fattah. Evaluation of a novel pegylated interferon alpha-2a (Reiferon Retard ®) in Egyptian patients with chronic hepatitis C – genotype 4. Digestive and Liver Disease. 2009; Supple 3:17– 19. M A Ahad, M A Alim and A R M Saifuddin Ekram. Interferon to PEG-Interferon: A Review. The Journal of Teachers Association. 2004 Dec;17(2):113-116. Mohammad Hanif Ghani, Naila Masood, Akrum Munir, Ghulam Hussain Baloch and Rafi Ahmed Ghori. Haematological disorders during interferon and ribavirin therapy in chronic hepatitis C patients. Medical Channel. 2009 October-December;15(4):167-70. M Schmid, A Kreil, W Jessner, M Homoncik, C Datz, A Gangl, P Ferenci, M Peck-Radosavljevic. Suppression of haematopoiesis during therapy of chronic hepatitis C with different interferon alfa mono and combination therapy regimens. Gut 2005;54:1014–1020. Martin D Green (Hoffmann-La Roche, Inc).Clinical Pharmacology Review of Peg-interferon alfa-2a: Center for Biologics Evaluation and Research, Food and drug administration, Nutley, New Jersey; 2000. BLA 103964 review. Patrick Marcellin, George K. K. Lau, Stefan Zeuzem, et al. Comparing the safety, tolerability and qualityof life in patients with chronic hepatitis B vs chronic hepatitis C treated with peginterferon alpha-2a. Liver International. 2008;477-85. Alaa Awad Taha, Ahmad El-Ray, Maged El-Ghannam and Bahaa Mounir. Efficacy and safety of a novel pegylated interferon alpha-2a in Egyptian patients with genotype 4 chronic hepatitis C. Canadian Journal of Gastroenterology. October 2010;24(10):597-602. Michael W Fried, Mitchell L. Shiffman, K. Rajender Reddy, Coleman Smith, George Marinos , Fernando L. Gonçales , Jr, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. The New England Journal of Medicine., 2002 Sep, 26;347:13:975-82. R. J Motzer, A. Rakhit, J. Thompson, H. Gurney, P. Selby, R. Figlin, et al. Phase II trial of branched peginterferon-α 2a (40 kDa) for patients with advanced renal cell carcinoma. Annals of Oncology. 2002;13:1799–1805. Peter Varunok, Eric Lawitz, Kimberly L Beavers, Gary Matusow, Ruby Leong, Nathalie Lambert, et al. Evaluation of pharmacokinetics, user handling, and tolerability of peginterferon alfa-2a (40 kDa) delivered via a disposable autoinjector device. Patient Preference and Adherence. Nov 23, 2011;5:587-99. Robert J. Motzer, Ashok Rakhit, Michelle Ginsberg, Karen Rittweger, Jacqueline Vuky, Richard Yu, Scott Fettner, and Leon Hooftman. Phase I trial of 40-kDa branched pegylated interferon alfa-2a for patients with advanced renal cell carcinoma. Journal of Clinical Oncology. 2001 March 1;19(5):1312-19.

661


ISSN 2249-1848

22. Bruce A. Luxon, Michael Grace, Diana Brassard and Ronald Bordens. Pegylated interferons for the treatment of chronic hepatitis C infection. Clinical Therapeutics. 2002;24(9):1363-83. 23. Stefan Zeuzem, S. Victor Feinman, Jens Rasenack, E. Jenny Heathcote, Ming-Yang La, Edward Gane, et al. Peginterferon alfa-2a in patients with chronic hepatitis C. The New England Journal of Medicine. 2000 December 7;343(23):1666-72. 24. Th Witthoft, B. Moller, K. H. Wiedmann, St. Mauss, R. Link, J. Lohmeyer, et al. Safety, tolerability and efficacy of peginterferon alpha-2a and ribavirin in chronic hepatitis C in clinical practice: The German Open Safety Trial. Journal of Viral Hepatitis. 2007 September;14:788-96. 25. Idrian Garcia-Garcia, Carlos A González-Delgado, Carmen M Valenzuela-Silva, Alina Díaz-Machado, Marisol Cruz-Díaz, Hugo Nodarse-Cuni, Orlando Pérez-Pérez, et al. Pharmacokinetic and pharmacodynamic comparison of two “pegylated” interferon alpha-2 formulations in healthy male volunteers: a randomized, crossover, double-blind study. BMC Pharmacology. 2010;10:15. 26. Stefan Zeuzem, Moisés Diago, Edward Gane, K. Rajender Reddy, Paul Pockros, Daniele Prati, et al. Peginterferon alfa-2a (40kDa) and ribavirin in patients with chronic hepatitis C and normal aminotransferase levels. Gastroenterology. 2004;127:1724-32.

662